Human respiratory syncytial virus (HRSV) is the leading viral cause of serious pediatric respiratory disease worldwide and an important cause of severe respiratory disease in the elderly where it can be associated with as much excess mortality as influenza virus. No effective vaccine is available. Maternal antibody does not confer solid immunity on neonates and natural infection provides only partial protection as frequent reinfection occurs throughout life. To address this major public health problem, our research program has focused on elucidating the molecular biology of HRSV with the goal of understanding not only the role of individual viral gene products in the infectious process but how expression of individual genes is controlled and how ability to alter the control of gene expression may affect the overall fitness of these viruses and their pathogenicity. Using reverse genetics to engineer changes into the HRSV genome we propose to examine the importance of transcriptional control in the viral life cycle and the role of individual gene products in the ability of the virus to assemble and mature infectious virus particles. This proposal describes our plans to investigate these central and poorly understood aspects of the viral life cycle. The specific aims are: 1. To analyze the importance of varied transcriptional termination efficiency and its impact on the transcriptional gradient in the HRSV life cycle and viral fitness. 2. To analyze transcriptional attenuation at the varied gene junctions and the features that contribute to it. 3. To investigate the role of the M2-1 protein and its modification on function in infection. 4. To analyze the functions of polymerase regions involved in transcription. 5.To analyze the physical structure of infectious HRSV particles. 6. To analyze the viral protein requirements for assembly, maturation and budding of virus particles using a trans-complementing cell line that allows deletion of one, two or all three of the surface glycoproteins, SH, G or F. 7. To analyze the viral components that target virus to distinct locations in vivo. [unreadable] [unreadable] [unreadable]